鼠源IL-1β基因的克

发布时间：2018-07-26 12:02

【摘要】： 类风湿性关节炎(RA)是一种以关节滑膜炎及对称性、破坏性的关节病变为主要特征的慢性全身性自身免疫性疾病。滑膜炎的持久反复发作,导致关节软骨及骨质破坏,最终导致关节畸型及功能障碍,血管炎可侵犯全身许多器官,引起系统性病变。半个世纪以来,人们提出多种假说来尝试解释RA,但是因为RA的发病机制过于复杂,临床上至今尚无有效的治疗药物,因此在临床上一直需要一种有效的治疗RA的药物。而研究并建立合理有效的类风湿性关节炎动物模型则是研制开发有效治疗药物和制订有效治疗措施的关键。白介素细胞-1β(IL-1β)是第一个从滑膜里发现的可以加速人体软骨破坏的细胞因子,虽然导致关节炎的病因目前并无定论。但已经有观点认为细胞因子在关节炎的长期病程中有其关键的作用,现在FDA已经批准用IL-1β的受体拮抗剂的基因方法治疗关节炎。RA是一个长期的缓慢的炎性因子介导的疾病过程,它的一个明显的特征就是不可控制的滑膜增生和丰富的炎性细胞浸润以及关节的侵蚀性浸润。在RA的发病机理中,细胞因子间的相互作用包括IL-1β,TNF-α和其它炎性因子。这些因子在激活成纤维因子和长期的关节及软骨破坏中共同起到了决定性的作用。除了参与机体免疫调节外,IL-1β在很多疾病中都发挥作用,特别是类风湿性关节炎。由于传统药物在治疗中所表现出的单一性和低效性,使人们开始倾向于开发一种能够抗细胞因子的一种新的方法来治疗类风湿关节炎。为更好的评价IL-1β抗体的药理和药效,也为了更深入的在动物实验上研究IL-1β的生物学特性,在借鉴人源IL-1β成功高效表达的基础上,本研究通过RT-PCR技术从LPS刺激过的小鼠胸腺细胞中成功克隆鼠源IL-1β成熟蛋白基因。通过pSUMO原核表达载体的重新构建,获得高效表达的具有生物活性的可溶性鼠源IL-1β蛋白,为深入研究和利用IL-1β奠定基础。本研究也成功建立了SD大鼠的鸡Ⅱ型胶原(鸡CII+CFA)关节炎动物模型。并通过一系列摸索研究,在原有模型成功建立的基础上将鸡Ⅱ型胶原与佐剂混合免疫大鼠结合腹腔注射鼠源IL-1β(鸡CII+CFA+IL-1β)制备新型的大鼠关节炎模型,这种模型从病程期上看要明显早于只注射鸡Ⅱ型胶原(鸡CII+CFA)的模型组,从炎症的严重程度上来看,也要明显高于鸡Ⅱ型胶原(鸡CII+CFA)的模型组,因此IL-1β可应用于建立RA模型,为研究RA的发病机制和研发治疗药物提供了一种新的建模方法。本研究ELISA的方法检测了所建立的模型动物血清中抗CII抗体、IL-1β、IL-17和TNF-α水平,结果表明模型动物血清中抗CII抗体、IL-1β、IL-17和TNF-α水平都有显著提高,与对照组相比差异显著(*P0.05)。以关节炎指数、抗CⅡ抗体滴度、细胞因子水平、组织病理变化和影像学变化作为评价指标,能够对大鼠关节炎模型做出较全面的综合评价。结果表明本方法极大地丰富了RA大鼠模型制备的途径。
[Abstract]:Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint synovitis and symmetrical, destructive joint lesions. The persistent recurrent attacks of synovitis lead to articular cartilage and bone destruction, resulting in joint malformation and dysfunction. Vasculitis can invade many organs of the body and cause the system. For half a century, people have put forward a variety of hypotheses to try to explain RA, but because the pathogenesis of RA is too complicated, there is no effective therapeutic drug in clinic so far, so an effective drug for the treatment of RA has been needed in clinical. And the research and establishment of a reasonable and effective animal model of rheumatoid arthritis is developed. The key to developing effective drugs and developing effective treatment measures.
Interleukin cell -1 beta (IL-1 beta) is the first cytokine that can accelerate the destruction of human cartilage from the synovial membrane. Although the cause of arthritis is not conclusive, there is a view that cytokine plays a key role in the long-term course of arthritis. Now, the base of IL-1 beta receptor antagonist has been approved in FDA. The treatment of arthritis.RA is a long-term, slow, inflammatory factor mediated disease process. It is characterized by an incontrollable synovial hyperplasia, rich inflammatory cell infiltration and erosive infiltration of the joints. In the pathogenesis of RA, the interaction of cytokines includes IL-1 beta, TNF- alpha and other inflammatory properties. Factors. These factors play a decisive role in activating fibroblasts and long-term joint and cartilage destruction. In addition to the immune regulation of the body, IL-1 beta plays a role in many diseases, especially rheumatoid arthritis. It is beginning to develop a new anti cytokine method to treat rheumatoid arthritis.
In order to better evaluate the pharmacology and efficacy of IL-1 beta antibody and to study the biological characteristics of IL-1 beta in animal experiments, on the basis of the successful and efficient expression of human IL-1 beta, this study successfully cloned the mouse source IL-1 beta mature protein gene from the mouse thymus stimulated by LPS by RT-PCR technology. Through pSUMO prokaryotic cells. The expression vector was rebuilt to obtain highly bioactive soluble mouse IL-1 beta protein, which lay the foundation for further research and utilization of IL-1 beta.
This study also successfully established an animal model of chicken type II collagen (chicken CII+CFA) arthritis in SD rats, and through a series of exploratory studies, a new model of rat arthritis was prepared by intraperitoneal injection of chicken type II collagen and adjuvant combined with IL-1 beta (chicken CII+CFA+ IL-1 beta) on the basis of the successful establishment of the original model. The model group is obviously earlier than the model group that only injected chicken type II collagen (chicken CII+CFA). From the severity of inflammation, it is also significantly higher than the model group of chicken type II collagen (chicken CII+CFA). Therefore, IL-1 beta can be used to establish the RA model, which provides a new modeling method for the study of the pathogenesis of RA and the research and development of drugs for the treatment of drugs. In this study, the ELISA method was used to detect the anti CII antibody, IL-1 beta, IL-17 and TNF- alpha levels in the model animal serum. The results showed that the levels of anti CII antibody, IL-1 beta, IL-17 and TNF- alpha in the serum of the model animals were significantly higher than those of the control group (*P0.05). The titer of anti C II antibody, the level of cytokines, the level of cytokines, and the level of cytokines, and the levels of cytokines, and the levels of cytokines, and the levels of cytokines, and the levels of cytokines, and the levels of cytokines, and cytokines, in the serum of the model animals were significantly higher than those in the control group. The pathological changes and imaging changes are used as evaluation indicators to make a comprehensive and comprehensive evaluation of the rat model of arthritis. The results show that this method greatly enriches the way of RA rat model preparation.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332;R593.22

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